Throughout and within this application various technical and patent literature are referenced either explicitly or by reference to an Arabic numeral. The bibliographic citations for the Arabic numeral citations are found after the experimental examples. The contents of these technical and patent citations are incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
To date, visualization of proteins inside cells has been addressed in two distinct fashions, each with its own considerable limitations: 1) by creating fusions with fluorescent proteins, and 2) by labeling the endogenous proteins with antibodies after killing the cell and immobilizing the cellular components.
Green fluorescent protein (GFP) and other fluorescent protein variants provide a way to visualize specific proteins in cells as GFP-target protein fusions. These protein fusions can be inserted into cells directly (e.g., by transfection or microinjection) or by complex genetic manipulations such as mouse knock-ins that enable replacement of the endogenous copy with the GFP-labeled copy. Direct insertion into cells by transfection often results in non-physiologic over-expression of protein that can alter both the target proteins subcellular localization and cellular morphology. Construction of knockin mice is labor- and time-intensive and offers no opportunity to label the target protein in a conformation, complex, or modification-selective fashion.
Antibodies can be useful for labeling and visualizing cellular components. This utility is derived from the fact that antibodies can selectively recognize biopolymers such as proteins, RNA, DNA, oligosaccharides, and complexes thereof. With regards to protein targeting, antibodies can recognize specific protein sequences (linear epitopes), protein surfaces (3-dimensional epitopes), protein conformations (e.g., activated vs. inactivated), protein complexes, and proteins bearing posttranslational modifications.
For visualization, however, antibodies are traditionally deployed by using fixed cells—dead cells that have had the membrane portion stripped off and the protein components fused together via addition of cross-linking reagents. Antibodies can also be utilized to label tissues that have been frozen and sectioned, destroying the original tissue.
Recently, it has been shown that antibodies can be microinjected into cells and used for visualization. However, this approach is suboptimal as it requires injection of every cell to be visualized, is transient, has limited dosage control over the injection, and because the antibodies themselves will gradually be inactivated/degraded by the reducing environment inside the cell and cellular protein turnover. Furthermore, the amount of antibody in the cell must match exactly the amount of target, something that is not currently possible, either the target is not sufficiently labeled, or there is a high level of background label.
Currently there is no method for directly degrading specific endogenous proteins. Current methods such as siRNA target mRNA and thus protein elimination is indirect. Drawbacks of siRNA include but are not limited to several issues. The first is the fact that in siRNA, protein is not degraded, but production of protein is blocked, and thus elimination of protein depends on the protein turnover rate. As a result elimination of more than half of the protein can take over 2 weeks. The second is that in siRNA, there can be “off target” effects that result in blocking protein production from unintended RNA templates. A third limitation is that siRNA targets all proteins encoded by particular mRNAs. Thus all mRNAs containing the same nucleotide sequence are affected. This limitation makes it impossible to target proteins with specific conformations (e.g., activated or inactivated forms of the protein) or specific post-translational modifications that have the same mRNA sequence. This limitation also may make it difficult to target/avoid various splice variants from a particular gene. With regards to oncogenes and oncogeneic mutations, siRNA methods are also limited in targeting specific sites that differ from the wildtype gene sequence by one or a small number of nucleotides.